The field of the invention is peptides useful for treatment of disorders of the digestive system, disorders of the eye and disorders associated with unwanted apoptosis.
Jxc3x8rgensen et al. (1982, Regulatory Peptides 3:231) describe a porcine pancreatic peptide, pancreatic spasmolytic peptide (PSP). PSP was found to inhibit xe2x80x9cgastrointestinal motility and gastric acid secretion in laboratory animal after parenteral as well as oral administration.xe2x80x9d It was suggested that xe2x80x9cif the results in animal experiments can be confirmed in man, PSP may possess a potential utility in treatment of gastroduodenal ulcer diseases.xe2x80x9d
pS2 is a small cysteine-rich protein which is expressed and secreted from human breast tumours. In addition, pS2 protein is expressed in normal stomach mucosa and in regenerative tissues in ulcerative diseases of the gastrointestinal tract (Rio et al., Cancer Cells, 1990, 2:269-74).
In a first aspect, the invention features a purified nucleic acid encoding an intestinal trefoil factor (ITF).
In preferred embodiments, the ITF is a mammalian ITF, preferably human, rat, bovine, mouse, monkey or porcine ITF. In another preferred embodiment, the purified nucleic acid encoding an ITF is present within a vector. In one embodiment, the ITF is in a monomer form. In another embodiment, two monomer ITF can be linked by a disulfide bond to form a dimer.
In a related aspect, the invention features a cell that includes a vector encoding an ITF.
In another related aspect, the invention features a substantially pure ITF. In a preferred embodiment, the polypeptide is detectably labelled. In a related aspect, the invention features a therapeutic composition that includes an ITF and a pharmacologically acceptable carrier.
In another aspect the invention features ITF variants.
In another aspect, the invention features a monoclonal antibody which preferentially binds (i.e., forms an immune complex with) an ITF. In a preferred embodiment, the monoclonal antibody is detectably labelled.
In a related aspect, the invention features a method for detecting human ITF in a human patient. The method includes the steps of contacting a biological sample obtained from the patient with a monoclonal antibody which preferentially binds ITF, and detecting immune complexes formed with the monoclonal antibody. In preferred embodiments the biological sample is an intestinal mucosal scraping, or serum.
In a related aspect, the invention features a method for treating digestive disorders in a human patient, which method involves administering to the patient a therapeutic composition that includes an ITF and a pharmacologically acceptable carrier. In one embodiment, a wild-type ITF protein, e.g., a human ITF protein (FIG. 6, SEQ ID NO:4), is used to treat a digestive disorder. A wild-type ITF protein is resistant to destruction in the digestive tract, and can be used for treatment of a digestive disorder such as a peptic ulcer disease, an inflammatory bowel disease, and can be used to protect the intestinal tract from injury caused by insults such as radiation injury or bacterial infection.
In another related aspect, the invention features a method for treating an eye disorder in a human patient, which method involves administering to the patient a therapeutic composition that includes an ITF protein and a pharmacologically acceptable carrier. An ITF protein and biologically active fragments or variants thereof can be used for the treatment of eye disorders such as a corneal ulcer, or an ocular inflammatory disease. An ITF and biologically active fragments or variants thereof can be used to treat corneal injury or lesion associated with corneal transplantation, lens implantation and other types of eye surgery. ITF can also be used to treat traumatic physical injury to the eye. The methods of the invention also include treating eye disorders with SP or pS2 protein, e.g., a human Sp or pS2 protein (FIG. 9, SEQ ID NO:14 and FIG. 10, SEQ ID NO:16), respectively. In addition, biologically active fragments or variants of SP or pS2 can be used to treat eye disorders. Any or all of the trefoil proteins can be administered to treat an eye disorder (see Sands, Annual Rev. Physiol 58:253-73). ITF or pS2 can be administered in monomer form or can be administered in a dimer form.
In yet another related aspect, the invention features a method for modulating apoptosis in a human patient, which method involves administering to the patient a therapeutic composition that modulates expression or activity of ITF and a pharmacologically acceptable carrier. The methods of the invention also include a method of modulating apoptosis by administering a therapeutic composition that modulates expression or activity of SP or pS2. In addition, biologically active fragments or variants of SP or pS2 can be used to modulate apoptotic disorders. Any or all of the trefoil proteins can be administered. ITF or pS2 can be administered in monomer form or can be administered in a dimer form.
In another aspect, the invention features a method for detecting binding sites for ITF in a patient. The method involves contacting a biological sample obtained from the patient with the factor, and detecting the factor bound to the biological sample as an indication of the presence of the binding sites in the sample. By xe2x80x9cbinding sites,xe2x80x9d as used herein, is meant any antibody or receptor that binds to an ITF protein, factor, or analog. The detection or quantitation of binding sites may be a useful reflection of abnormalities of the digestive tract.
In another aspect, the invention features substantially pure ITF. In preferred embodiments, the ITF is human, porcine, mouse, rat, guinea pig, monkey, or bovine trefoil factor.
By xe2x80x9cintestinal trefoil factorxe2x80x9d (xe2x80x9cITFxe2x80x9d) is meant any protein that is substantially homologous to rat ITF (FIG. 2, SEQ ID NO.:2) or human ITF (FIG. 6, SEQ ID NO:4) and which is expressed in the large intestine, small intestine, or colon to a greater extent than it is expressed in tissues other than the small intestine, large intestine, or colon. Also included are: allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridizes under high or low stringency conditions to ITF encoding nucleic acids retrieved from naturally occurring material; and polypeptides or proteins retrieved by antisera to ITF, especially by antisera to the active site or binding domain of ITF. The term also includes other chimeric polypeptides that include an ITF.
The term ITF also includes analogs of naturally occurring ITF polypeptides. Analogs can differ from the naturally occurring ITF by amino acid sequence differences or by modifications that do not affect sequence, or by both. Analogs of the invention will generally exhibit at least 70%, more preferably 80%, more preferably 90%, and most preferably 95% or even 99%, homology with all or part of a naturally occurring ITF sequence. The length of comparison sequences will generally be at least 8 amino acid residues, usually at least 20 amino acid residues, more usually at least 24 amino acid residues, typically at least 28 amino acid residues, and preferably more than 35 amino acid residues. Modifications include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps, e.g., by exposing the polypeptide to enzymes that affect glycosylation derived from cells that normally provide such processing, e.g., mammalian glycosylation enzymes. Also embraced are versions of the same primary amino acid sequence that have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine. Analogs can differ from naturally occurring ITF by alterations of their primary sequence. These include genetic variants, both natural and induced. Induced mutants may be derived by various techniques, including random mutagenesis of the encoding nucleic acids using irradiation or exposure to ethanemethylsulfate (EMS), or may incorporate changes produced by site-specific mutagenesis or other techniques of molecular biology. See, Sambrook, Fritsch and Maniatis (1989), Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, hereby incorporated by reference. Also included are analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., xcex2 or xcex3 amino acids.
In addition to substantially full-length polypeptides, the term ITF, as used herein, includes A biologically active fragments of the polypeptides. As used herein, the term xe2x80x9cfragment,xe2x80x9d as applied to a polypeptide, will ordinarily be at least 10 contiguous amino acids, typically at least 20 contiguous amino acids, more typically at least 30 contiguous amino acids, usually at least 40 contiguous amino acids, preferably at least 50 contiguous amino acids, and most preferably at least 60 to 80 or more contiguous amino acids in length. Fragments of ITF can be generated by methods known to those skilled in the art and described herein. The ability of a candidate fragment to exhibit a biological activity of ITF can be assessed by methods known to those skilled in the art and are described herein. Also included in the term xe2x80x9cfragmentxe2x80x9d are biologically active ITF polypeptides containing amino acids that are normally removed during protein processing, including additional amino acids that are not required for the biological activity of the polypeptide, or including additional amino acids that result from alternative mRNA splicing or alternative protein processing events.
An ITF polypeptide, fragment, or analog is biologically active if it exhibits a biological activity of a naturally occurring ITF, e.g., the ability to alter gastrointestinal motility in a mammal or the ability to enhance corneal epithelial wound healing.
The invention also includes nucleic acid sequences, and purified preparations thereof, that encode the ITF polypeptides described herein. The invention also includes antibodies, preferably monoclonal antibodies, that bind specifically to ITF polypeptides.
ITF, SP and pS2 are referred to as trefoil proteins and are members of the trefoil family. These proteins are designated trefoil proteins because they have a trefoil shaped secondary structure which is stabilized by intrachain disulfide bonds. Members of the trefoil family typically will have at least one of the following properties in common: (i) a common structural domain, e.g., the trefoil shaped secondary structure, (ii) a degree of amino acid or nucleotide sequence homology, or (iii) a common functional characteristic.
Members of the trefoil family, e.g., ITF, are used in the treatments discussed above. Skilled artisans may review these proteins in Sands et al. (1996, Ann. Rev. Physiol. 58:253-273). As stated above, the invention encompasses biologically active fragments of the trefoil proteins. Fragments that retain the trefoil structure (i.e., the three loop structure) or that lie within regions of the protein that are highly conserved may prove particularly useful. Thus, portions of ITF, pS2, or SP from about the first cystine involved in a disulfide bond of the three loop structure to about the last cysteine involved in a disulfide bond of the three loop structure are useful. Biologically active fragments of ITF, pS2, SP can include 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 129 amino acids.
Variants of a selected trefoil protein are least 60%, preferably at least 75%, more preferably at least 90%, and most preferably at least 95% identical to the selected trefoil protein, preferably a human trefoil protein, more preferably human ITF.
The term xe2x80x9cidentical,xe2x80x9d as used herein in reference to polypeptide or DNA sequences, refers to the subunit sequence identity between two molecules. To determine the percent sequence identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions (e.g., overlapping positions)xc3x97100). In one embodiment the two sequences are the same length.
The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.
An isolated nucleic acid molecule encoding a xe2x80x9cvariant proteinxe2x80x9d can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of ITF, pS2 or SP such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A xe2x80x9cconservative amino acid substitutionxe2x80x9d is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
In the case of amino acid sequences that are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Sequence identity is typically measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin (Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705), and the default parameters specified therein.
A variant of a selected trefoil protein preferably has the amino acids present in the naturally-occurring form of the selected trefoil protein at the more highly conserved amino acid positions of the protein. Thus, a variant of human ITF preferably is identical to naturally-occurring human ITF at all or nearly all of the more highly conserved positions. Sequence conservation among trefoil proteins is evident in Table 1 of Sands et al. (supra) which can be used by those skilled in the art to identify more conserved residues. In one embodiment, a human SP variant polypeptide is identical to wild-type SP polypeptide at 129 (127, 125, 123, 121, 119, 117, or 115) of the 130 amino acid residues of the wild-type SP. Preferably the cysteine residues and the trefoil structure is preserved. The remaining amino acids can be replaced by conservative substitutions. In another embodiment, a human pS2 variant polypeptide is identical to wild type pS2 at 83 (81, 79, 77, 75, 73, 72, or 71) of the 84 amino acid residues of the wild-type pS2. Preferably the cysteine residues and the trefoil structure is preserved. The remaining pS2 amino acid residues can be replaced by conservative substitutions. In yet another embodiment, a human ITF variant polypeptide is identical to wild-type human ITF at 72 (71, 69, 67, 65, 63, 61 or 59) of the 73 amino acid residues of the wild-type human ITF. Preferably the cysteine residues and the trefoil structure is preserved. The remaining amino acids can be replaced by conservative substitutions.
As used herein, the term xe2x80x9csubstantially purexe2x80x9d describes a compound, e.g., a nucleic acid, a protein, or a polypeptide, e.g., an ITF protein or polypeptide, that is substantially free from the components that naturally accompany it. Typically, a compound is substantially pure when at least 60%, more preferably at least 75%, more preferably at least 90%, and most preferably at least 99%, of the total material (by volume, by wet or dry weight, or by mole per cent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., in the case of polypeptides, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
By xe2x80x9cisolated DNAxe2x80x9d is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the given DNA of the invention is derived, flank the DNA. The term xe2x80x9cisolated DNAxe2x80x9d thus encompasses, for example, cDNA, cloned genomic DNA, and synthetic DNA. A xe2x80x9cpurified nucleic acidxe2x80x9d, as used herein, refers to a nucleic acid sequence that is substantially free of other macromolecule (e.g., other nucleic acids and proteins) with which it naturally occurs within a cell. In preferred embodiments, less than 40% (and more preferably less than 25%) of the purified nucleic acid preparation consists of such other macromolecule.
xe2x80x9cHomologousxe2x80x9d, as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules, or two polypeptide molecules. When a subunit position in both of the molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half, e.g., 5 of 10, of the positions in two compound sequences are homologous then the two sequences are 50% homologous; if 90% of the positions, e.g., 9 of 10, are matched or homologous the two sequences share 90% homology. By way of example, the DNA sequences 3xe2x80x2ATTGCCxe2x80x25 and 3xe2x80x2TATGGCxe2x80x25 share 50% homology. By xe2x80x9csubstantially homologousxe2x80x9d is meant largely but not wholly homologous.
xe2x80x9cTreatment of lesionsxe2x80x9d encompasses both the inhibition of the formation of lesion and the healing of lesions already formed. Biologically active fragments and variants of a trefoil protein, particularly ITF, which promote healing of lesions, e.g., eye lesion, intestinal lesion, or inhibit the formation of lesions,e.g., eye lesions or intestinal lesions, are useful in the treatments of the invention.
xe2x80x9cDisorders of the eyexe2x80x9d refers to any disturbance, defect, or abnormality in eye function or structure (e.g., the eye disorder is a disorder resulting from a disruption of the corneal epithelium). An eye disorder can be congenital, hereditary, or can be the result of a trauma such as a physical injury, an illness, inflammation, an autoimmune disease, a virus (e.g., adenoviruses, herpes simplex virus), a blepharitis, a keratitis sicca, a trachoma, a corneal foreign body, ultraviolet light exposure (e.g., welding arcs, sunlamps), contact lens overwear, a systemic drug (e.g., adenine arabinoside), a tropical drug, or invasion by a microbe.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.